'bowtie2' is not recognized.
 

Hi all,

I'm currently trying to use bowtie2 to map our RNAseq data to the mRNA reference / chromosomes reference. Building indexes went ok, however when trying to use bowtie2 -p 1 -x [path to 6.bt indexes] [fastq file] > [sam file] it says 'bowtie2' is not recognized as an internal or external command, operable program or batch file.

I'm using the cmd on windows. Anyone who can help me out here?

You might have to type the full PATH to bowtie2. As an aside, it's going to be a lot of hassle to deal with bioinformatics programs on windows (try to borrow someone's Mac).

Also, unless your organism doesn't have much splicing, you'll probably get better results using tophat2.

Ah it worked now, doing bowtie2-align instead of bowtie2. Anyhow, I was trying to map 60 homo sapiens mRNA sequences against the indexed reference mRNA of the homo sapiens, and 100.00% aligned 0 times.

Is this normal? O_O

Probably not, but I'd have to see the reads and know more about how they were generated to tell you for sure. Try using the "--local" option to see if that changes things. Also, did you quality/adapter trim your reads at all?

Reads were generated with Illumina MiSeq 2 paired-end reading. Adapters & Barcodes were trimmed off.

I'm using command bowtie-align -x hg19 -1 [file1] -2 [file2] -S [output] -fr -q

Using extra parameter --local generates 92.50% aligned 0 times.

You might try quality trimming things to see if that solves the problem. Alternatively, try blasting a few reads and see how they align. It may be that you simply need to change the defaults for bowtie (e.g., score-min).